PROJECT SUMMARY Type 1 diabetes (T1D) is an autoimmune disease that affects millions of people worldwide. The incidence of T1D is rising, especially in young children. Although significant progress has been made to predict who is at risk for developing T1D, there are no effective therapies to prevent this disease. Both genetic and environmental factors contribute to the risk of developing T1D. Certain human leukocyte antigen (HLA) haplotypes dominantly protect against the development of T1D, yet the mechanism of this remarkable protection from autoimmunity is not well-understood. NOD mice, the most widely used model of T1D, do not express a major histocompatibility complex (MHC) class II E molecule. Transgenic expression of the MHCII E molecule in NOD mice (E?16/NOD) completely prevents T1D, mirroring dominant HLA protection from T1D in humans. Using these E?16/NOD mice as a model of dominant genetic protection from T1D, we recently demonstrated that MHC/HLA genetic protection from autoimmunity operates via immune system selection of diabetes-protective commensal microbiota early in life, which in turn, influences the developing immune system. Since we find an increased proportion of intestinal Tregs and a distinct early-life microbiome in Ea16/NOD mice, our central hypothesis is that specific microbes prevent T1D by promoting development of diabetes-protective CD4+ regulatory T cells (Tregs). Modeling of HLA class II dominant protection from T1D in murine models may provide critical insights to support our long-term goal of developing microbiota-based therapies to prevent T1D in humans. Due to the complexity and high levels of variability of the intestinal microbiome, determining the specific microbial strains that educate the immune system is problematic. Building upon established techniques of microbial flow cytometry of antibody-coated commensal microbes, we developed an innovative approach called mFLOW-SEQ to identify microbes that stimulate the CD4+ T cell compartment in a model of genetic protection from T1D. Aim 1 leverages our innovative mFLOW- SEQ approach to identify commensal microbes that induce CD4+ Treg cells and prevent T1D. Aim 2 uses genetic models to determine the extent to which early-life Treg cells prevent autoimmunity in genetically protected E?16/NOD mice. Successful completion of these aims will provide critical information on which early- life microbes impact the development of the immune system to prevent T1D and further whether early-life Tregs provide protection from T1D. In addition, mFLOW-SEQ is an innovative tool for identifying microbes that modulate the immune system in mice. As a future direction, we envision applying mFLOW-SEQ to human samples to help generate personalized microbiota therapies based on HLA haplotypes for human patients at risk for T1D.